LRRC8 is a Leucine Rich Repeat Containing transmembrane protein expressed on the surface of both hematopoietic and non-hematopoietic cells. The biological function of LRRC8 and the identity of its ligand(s) are unknown. A heterozygous mutation in LRRC8 has recently been described in an immunodeficient patient with agammaglobulinemia. The mutant was thought to act as a dominant negative because murine CD34+ progenitors that express the mutant protein had impaired B and T cell development in radiation chimera mice. A role for LRRC8 in the development of T and B lymphocytes was firmly established by our observation that LRRC8-/- mice have drastically decreased number of thymocytes (10% of normal) with a severe block in transition from the DN to the DP stage. LRRC8-/- peripheral T cells were severely impaired in their ability to proliferate in response to TCR stimulation even after addition of exogenous IL-2. Our recent analysis of RAG2 deficient mice reconstituted with LRRC8-/- fetal liver cells has unequivocally established a cell intrinsic role for LRRC8 in T cell development and function. These results strongly suggest that LRRC8 is essential for normal T cell development and function. The objective of our proposal is to establish a role for LRRC8 in T cell activation and to identify its ligand(s). Our preliminary results indicate that LRRC8 monomers assemble as homotrimers in transfected T293 cells, that LRRC8 associate intra-cellularly with Grb2, and that ligation of LRRC8 results in activation of the MAP kinases Erk, JNK and p38. We hypothesize that following binding to its endogenous ligand(s) LRRC8 activates T cells and that this is critical for their normal development and function. We also hypothesize that the LRRC8 truncation mutant of the patient disrupts LRRC8 signaling by assembling with WT LRRC8. To test our hypothesis, we propose to examine LRRC8 mediated signaling in T cells using LRRC8 agonist mAb and to search for the endogenous LRRC8 ligand(s). We anticipate that the results obtained from our proposed study will define the role of LRRC8 in T cell activation and shed light on the pathogenesis of LRRC8 deficiency. PUBLIC HEALTH RELEVANCE: A mutation in a novel gene LRRC8 (leucine rich repeat containing 8) has recently been described in an immunodeficient patient with agammaglobulinemia. Our preliminary studies, using LRRC8 deficient mouse model, have strongly suggested a critical role for LRRC8 in T cell development and function. The objectives of our proposal are to investigate the role of LRRC8 as a surface receptor that delivers an activating signal to T cells, and to identify its endogenous ligand(s). [unreadable] [unreadable] [unreadable]